QuantiFast Probe PCR Kits deliver fast and sensitive quantification of gDNA and cDNA targets by real-time PCR and two-step RT-PCR using sequence-specific probes. Q-bond technology and an optimized master mix enable shorter real-time PCR run times, not only on fast cyclers with short ramping times, but also on standard cyclers. The combination of a hot start and a unique PCR buffer system in the ready-to-use master mix ensures highly sensitive qPCR on any real-time cycler without the need for optimization. Two kit formats are available: the QuantiFast Probe PCR Kit for cyclers that require ROX dye for fluorescence normalization, and the QuantiFast Probe PCR +ROX Vial Kit for all other cyclers. For convenience, the master mix can be stored at 2–8°C.

QuantiFast Probe Kits reduce total PCR run time by up to 60% in real-time two-step RT-PCR on standard cyclers (40 PCR cycles carried out; comparison with a standard QIAGEN real-time PCR kit). L: LightCycler 2.0; A1: ABI PRISM 7900; A2: Applied Biosystems 7500; A3: ABI PRISM 7000. |The QuantiFast Probe PCR +ROX Vial Kit provided accurate gene expression analysis from low to high template amounts with a PCR efficiency of 93% in two-step PCR on the Mastercycler ep realplex. Duplicate reactions (25 μl volume) were performed using 10-fold dilutions of human leukocyte cDNA and a Primer Express designed TaqMan assay for ubiquitin (a regulatory protein). NTC: no template control.|Reactions were run in duplicate using 10-fold dilutions of human leukocyte cDNA (100 ng to 0.01 ng) and a TaqMan gene expression assay for IL1RN (a cytokine) on the ABI PRISM 7900. The QuantiFast Kit showed greater sensitivity than the standard cycling kit from Supplier AII (which was used according to the standard cycling protocol), providing much lower CT values and transcript quantification from down to 10 pg cDNA.|Reactions were run in triplicate using 10-fold dilutions of human leukocyte cDNA (100 ng to 0.01 ng) and a Primer Express designed TaqMan assay for IL8 (a chemokine) on the ABI PRISM 7900. [A] The QuantiFast Kit showed greater sensitivity than [B] the kit from Supplier I (which was used according to the standard cycling protocol), providing lower CT values and transcript quantification from down to 0.01 ng cDNA.|Duplicate two-step RT-PCR reactions were run using a set of primers and FRET probes for b-2 microglobulin from Supplier R on the LightCycler 2.0. The amplification plots for the QuantiFast Kit show the kit's wide dynamic range.|A balanced combination of KCl and (NH4)2SO4 promotes specific annealing of primers and probes to the PCR template. K+ binds to phosphate groups on double-stranded DNA, stabilizing annealing of primers and probes. NH4+ destabilizes weak hydrogen bonds between mismatched bases.|[A] Q-Bond in QuantiFast Buffer increases the affinity of DNA polymerase for short single-stranded DNA, reducing primer annealing time to a few seconds. In addition, the unique buffer composition supports the melting of DNA, reducing denaturation and extension times. [B] Without Q-Bond, the primer and polymerase bind sequentially to the template, increasing primer annealing time.|

Triplicate reactions were run using a Primer Express designed TaqMan® assay for IL8 (a chemokine). The QuantiFast Kit provided lower CT values than the kit from Supplier BV (which was used according to a fast-cycling protocol), allowing transcript quantification from just 1 ng cDNA.

Detection over 7 logs of template in two-step RT-PCR on the LightCycler 2.0

Mean CT value

Human leukocyte cDNA

QuantiFast Probe PCR +ROX Vial Kit

Kit from Supplier R

100 ng

11.99

14.56

10 ng

15.54

17.99

1 ng

19.22

22.16

100 pg

22.63

25.14

10 pg

26.43

30.15

1 pg

30.02

34.43

100 fg

33.42

37.10

No template control

40

40.00

PCR efficiency

90%

80.1%

Duplicate reactions were run using a set of primers and FRET probes for β-2 microglobulin from supplier R. The QuantiFast Kit provided lower CT values and higher PCR efficiency that the kit from Supplier R (which was used according to the fast cycling protocol ).

Principle

QuantiFast Probe PCR Kits deliver highly sensitive and rapid results over a wide dynamic range on both standard and fast cyclers. The kits are designed for use with all types of sequence-specific probes, including hydrolysis probe detection (e.g., TaqMan and other dual-labeled probes) and FRET probes. A specially developed, fast PCR buffer contains the novel additive Q-Bond, which significantly reduces denaturation, annealing, and extension times (see figure "Fast primer annealing"). The buffer also contains a balanced combination of K+ and NH4+ ions, which promote specific primer annealing, enabling high PCR specificity and sensitivity (see figure "Specific primer annealing"). In addition, HotStarTaq Plus DNA Polymerase provides a stringent hot start, preventing the formation of nonspecific products.

Components of 2x QuantiFast Probe PCR Kit*

Component

Features and benefits

Benefits

HotStarTaq Plus DNA Polymerase

3 min activation at 95ºC

Set up of qPCR reactions at room temperature

QuantiFast Probe PCR Buffer

Balanced combination of NH4+ and K+ ions

Specific primer annealing ensures reliable PCR results

Unique Q-Bond additive

Faster PCR run times, enabling faster results and more reactions per day

Precise quantification on cyclers that require ROX dye. Does not interfere with PCR on any real-time cycler

* Also contains dNTP mix (dATP, dCTP, dGTP, and dTTP). † ROX dye is either present in the master mix or as a separate solution.

Procedure

QuantiFast Probe PCR Kits contain ready-to-use master mixes that eliminate the need for optimization of reaction and cycling conditions. Simply add template DNA, primers, and probe to the master mix and follow the protocol in the handbook to get fast and reliable results on any real-time cycler. Kits are available with or without ROX passive reference dye in the master mix, enabling use on virtually any real-time cycler (see table). Due to the optimized ROX concentrations, detection of even low copy numbers is achieved through automatic data analysis.

QuantiFast Probe Assays are predesigned, genomewide assays that use hydrolysis, probe-based detection. They are delivered with the QuantiFast Probe PCR Kit for guaranteed results in singleplex, two-step qRT-PCR.

Applications

QuantiFast Probe PCR Kits can be used for probe-based gene expression analysis of cDNA targets and quantitative gDNA analysis on any real-time cycler. This includes instruments from Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, Roche, and Agilent. For the Rotor-Gene Q and other Rotor-Gene cyclers, we recommend using the Rotor-Gene Probe PCR Kit, which has been specially developed for fast cycling on these instruments.